Kohler and Milstein are generally credited with having devised the techniques that successfully resulted in the formation of the first monoclonal antibody-producing hybridomas (G. Kohler and C. Milstein (1975) Nature 256:495-497; (1976), Eur. J. Immunol. 6:511-519). By fusing antibody-forming cells (spleen B-lymphocytes) with myeloma cells (malignant cells of bone marrow primary tumors), they created a hybrid cell line arising from a single fused cell hybrid (called a hybridoma or clone). The hybridoma had inherited certain characteristics of both the lymphocytes and the myeloma cell lines. Like the lymphocytes, the hybridoma secreted a single type of immunoglobulin; moreover, like the myeloma cells, the hybridoma had the potential for indefinite cell division. The combination of these two features offered distinct advantages over conventional antisera.
Antisera derived from vaccinated animals are variable mixtures of polyclonal antibodies which never can be reproduced identically. Monoclonal antibodies are highly specific immunoglobulins of a single type. The single type of immunoglobulins secreted by a hybridoma is specific to one and only one antigenic determinant, or epitope, on the antigen, a complex molecule having a multiplicity of antigenic determinants. For instance, if the antigen is a protein, an antigenic determinant may be one of the many peptide sequences (generally 6-7 amino acids in length; Atassi, M. Z. (1980) Molec. Cell. Biochem. 32:21-43) within the entire protein molecule. Hence, monoclonal antibodies raised against a single antigen may be distinct from each other depending on the determinant that induced their formation. For any given hybridoma, however, all of the antibodies it produces are identical. Furthermore, the hybridoma cell line is easily propagated in vitro or in vivo, and yields monoclonal antibodies in extremely high concentration.
A monoclonal antibody can be utilized as a probe to detect its antigen. Thus, monoclonal antibodies have been used in in vitro diagnostics, for example, radioimmunoassays and enzyme-linked immunoassays (ELISA), and in in vivo diagnostics, e.g. in vivo imaging with a radio-labeled monoclonal antibody. Also, a monoclonal antibody can be utilized as a vehicle for drug delivery to such antibodies' antigen.
Before a monoclonal antibody can be utilized for such purpose, however, it is essential that the monoclonal antibody be capable of binding to the antigen of interest; i.e., the target antigen. This procedure is carried out by screening the hybridomas that are formed to determine which hybridomas, if any, produce a monoclonal antibody that is capable of binding to the target antigen. This screening procedure can be very tedious in that numerous, for example, perhaps several thousand monoclonal antibodies may have to be screened before a hybridoma that produces an antibody that is capable of binding the target antigen is identified. Accordingly, there is a need for a method for the production of monoclonal antibodies that increases the likelihood that the hybridoma will produce an antibody to the target antigen.
Additionally, the immune systems of conventional animals used in the production of monoclonal antibodies cannot recognize epitopes that are highly conserved among vertebrate, and particularly mammalian species, as “non-self” because of “self” tolerance. The term “tolerance” is well known in the art and refers to the failure of an animal's immune system to respond to its own tissues. To the animal's immune system, a highly conserved epitope appears to be “self”, and no immune response is generated. Therefore, conventional animals are ineffective in the production of antibodies against such highly conserved epitopes.
There have been attempts in the prior art to address the problems found in the production of monoclonal antibodies, particularly with respect to the streamlining of the screening process for monoclonal antibodies and, to a certain extent, to the generation of a monoclonal antibody to an epitope that is highly conservative among animal species, particularly mammalian species.
One such attempt is described in U.S. Pat. No. 5,223,410 issued to Gargan et al. on Jun. 29, 1993, assigned to American Biogenetic Sciences, Inc. This patent describes a method for producing antibodies using an antigen-free animal. Particularly, it describes the production of monoclonal antibodies using sterile or germ-free mice. This patent focuses on the problem of streamlining of the screening processes for monoclonal antibodies by providing antigen-free or germ-free animals in which monoclonal antibodies can be more easily identified.
Korth et al. (1997) “Prion (PrPSc)-Specific Epitope Defined by a Monoclonal Antibody” (Letter to Nature) Nature 390:74 describes a monoclonal antibody, 15B3, that can discriminate between the normal and disease-specific forms of a prion (PrP). Prions are infectious particles causing transmissible spongiform encephalopathies. 15B3 specifically precipitates bovine, murine, or human PrPSc (the disease causing form), but not PrPc (the normal form), suggesting that it recognizes an epitope common to prions from different species. The 15B3 epitope was mapped as three polypeptide segments in PrP using immobilized synthetic peptides. However, the biological activity of this monoclonal antibody, which was produced from BALB/c mice, is uncharacterized.
In light of the above, a need exists for a method for making monoclonal antibodies against epitopes that are highly conservative among vertebrate, and particularly, mammalian species.